The aim of this study was to conduct an investigation into the bacterial diversity in the freshwater-deprived Kariega Estuary, situated along the Eastern Cape coastline, using ribosomal RNA gene sequences obtained by pyrosequencing. Shifts in the microbial diversity were correlated to selected physico-chemical variables along the length of the estuary. More than 27 000 sequences were obtained and rarefaction analyses confirmed a comprehensive appraisal of the microbial diversity present in the Kariega Estuary. Distinct patterns in phylotype distribution from the hypersaline upper reaches to the mouth of the estuary were observed; notably, the importance of the detrital food web within the Kariega Estuary was highlighted by the high occurrence of Bacteroidetes and Actinomycetes. Moreover, while the observed chlorophyll-a concentrations were low (< 0.1 µg·ℓ-1), the presence of Pelagibacter and Flavobacteria amongst the microbial community suggests a potentially important contribution of these microbes towards the total primary productivity of the ecosystem. No human pathogenic microbes were detected within waters of the system. We conclude that pyrosequencing provides a versatile and efficient tool for assessing the microbial diversity in the Kariega Estuary and propose that this technology may provide valuable information on the ecosystem functioning and health of aquatic ecosystems.